Recombinant DNA technology comprises extremely varied and powerful molecular biology techniques aimed at modifying nucleic acids at the DNA level and makes it possible to analyze and modify genomes at the molecular level. In this respect, viruses because of the small size of their genome are particularly amenable to such manipulations. However, recombinant DNA technology is not immediately applicable to nonretroviral RNA viruses because these viruses do not encompass a DNA intermediate step in their replication. For such viruses infectious clones (for instance as DNA copy or as in vitro transcribed RNA copy or as derivative of either) have to be developed before recombinant DNA technology can be applied to their genome to generate modified virus. Infectious clones can be derived through the construction of full-length (genomic length) cDNA (here used in the broad sense of a DNA copy of RNA and not only in the strict sense of a DNA copy of mRNA) of the virus under study after which an infectious transcript is synthesized in vivo in cells transfected with the full-length cDNA, but infectious transcripts can also be obtained by in vitro transcription from in vitro ligated partial-length cDNA fragments that comprise the full viral genome. In all cases, the transcribed RNA carries all the modifications that have been introduced to the cDNA and can be used to further passage the thus modified virus.
Infectious cDNA clones and infectious in vitro transcripts have been generated for a great number of positive strand RNA viruses (for a review see Boyer and Haenni, Virology 198, 415-426) with a genome of up to 12 kb or slightly larger. The viral genomic length of Pestiviruses seems until now the longest positive strand viral RNA genome from which infectious clones (Moormann et al., J. Vir. 70:763-770) have been prepared. Problems associated with genomic length lie not only in the difficulty of obtaining and maintaining long and stabile cDNA clones in bacteria but also in the infectivity of the initial RNA transcript of which replication in the host cell has to be achieved without the help of the normally associated viral proteins connected with viral replication. To achieve successful infection, viral transcripts must interact with viral-encoded proteins, most particularly with the viral replicase and with host cell components such as the translation machinery; therefore, the structure of viral transcripts has to mimic that of virion RNA as closely as possible. Additional problems can be found with those positive strand RNA viruses that replicate via a mechanism of subgenomic messenger RNAs transcribed from the 3' side of the genome and with those positive strand RNA viruses that generate during replication defective interfering particles, such as naked capsids or empty shell particles, comprising several structural proteins but only a part of the genome. The presence of incomplete viral RNA fragments or of e.g. matrix or nucleocapsid proteins interacting or interfering with the viral RNA to be transcribed or to replicative intermediate RNA and disrupting its structure will abolish full-length RNA strand synthesis, and thus the generation of infectious virus comprising genomic length RNA.
Lelystad virus (LV), also called porcine reproductive respiratory syndrome virus (PRRSV, genomic length 15.2 kb), is a member of the family Arteriviridae, which also comprises equine arteritis virus (EAV, genomic length 12.7 kb), lactate dehydrogenase-elevating virus (LDV, genomic length at least 14.2 kb) and simian hemorrhagic fever virus (SHFV genomic length approximately 15 kb) (Meulenberg et al., 1993a; Plagemann and Moennig, 1993).
Recently, the International Committee on the Taxonomy of Viruses has decided to incorporate this family in a new order of viruses, the Nidovirales, together with the Coronaviridae (genomic length 28 to 30 kb), and Toroviridae (genomic length 26 to 28 kb). The order Nidovirales represents enveloped RNA viruses that contain a positive-stranded RNA genome and synthesize a 3' nested set of subgenomic RNAs during replication. The subgenomic RNAs of coronaviruses and arteriviruses contain a leader sequence which is derived from the 5' end of the viral genome (Spaan et al., 1988; Plagemann and Moennig, 1993). The subgenomic RNAs of toroviruses lack a leader sequence (Snijder and Horzinek, 1993). Whereas the ORFs 1a and 1b, encoding the RNA dependent RNA polymerase, are expressed from the genomic RNA, the smaller ORFs at the 3' end of the genomes of Nidovirales encoding structural proteins are expressed from the subgenomic mRNAs.
PRRSV (Lelystad virus) was first isolated in 1991 by Wensvoort et al. (1991) and was shown to be the causative agent of a new disease now known as porcine reproductive respiratory syndrome (PRRS). The main symptoms of the disease are respiratory problems in pigs and abortions in sows. Although the major outbreaks, such as observed at first in the US in 1987 and in Europe in 1991, have diminished, this virus still causes economic losses in herds in the US, Europe, and Asia. PRRSV preferentially grows in alveolar lung macrophages (Wensvoort et al., 1991). A few cell lines, such as CL2621 and other cell lines cloned from the monkey kidney cell line MA-104 (Benfield et al., 1992; Collins et al., 1992; Kim et al., 1993), are also susceptible to the virus. Some well known PRRSV strains are known under accession numbers CNCM I-1102, I-1140, I-1387, I-1388, ECACC V93070108, or ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402. The genome of PRRSV was completely or partly sequenced (Conzelmann et al., 1993; Meulenberg et al., 1993a, Murthaugh et al, 1995) and encodes, besides the RNA dependent RNA polymerase (ORFs 1a and 1b), six structural proteins, of which four envelope glycoproteins named GP.sub.2 (ORF2), GP.sub.3 (ORF3), GP.sub.4 (ORF4) and GP.sub.5 (ORF5), a nonglycosylated membrane protein M (ORF6) and the nucleocapsid protein N (ORF7) (Meulenberg et al. 1995, 1996; van Nieuwstadt et al., 1996). Immunological characterization and nucleotide sequencing of EP and US strains of PRRSV has identified minor antigenic differences within strains of PRRSV located in the structural viral proteins (Nelson et al., 1993; Wensvoort et al., 1992; Murtaugh et al., 1995).
Pigs can be infected by PRRSV via the oronasal route. Virus in the lungs is taken up by lung alveolar macrophages and in these cells replication of PRRSV is completed within 9 hours. PRRSV travels from the lungs to the lung lymphnodes within 12 hours and to peripheral lymphnodes, bone marrow and spleen within 3 days. At these sites, only a few cells stain positive for viral antigen. The virus is present in the blood during at least 21 days and often much longer. After 7 days, antibodies to PRRSV are found in the blood. The combined presence of virus and antibody in PRRS infected pigs shows that the virus infection can persist for a long time, albeit at a low level, despite the presence of antibody. During at least 7 weeks, the population of alveolar cells in the lungs is different from normal SPF lungs.
PRRSV needs its envelope to infect pigs via the oronasal route and the normal immune response of the pig thus entails, among others, the production of neutralising antibodies directed against one or more of the envelope proteins; such antibodies can render the virus non-infective. However, once in the alveolar macrophage, the virus also produces naked capsids, constructed of RNA encapsidated by the M and/or N protein, sometimes partly containing any one of the glycoproteins. The intra- and extracellular presence of these incomplete viral particles or (partly) naked capsids can be demonstrated by electron microscopy. Sometimes, naked capsids without a nucleic acid content can be found. The naked capsids are distributed through the body by the bloodstream and are taken up from the blood by macrophages in spleen, lymphnodes and bone marrow. These naked but infectious viral capsids can not be neutralised by the antibodies generated by the pig and thus explain the persistence of the viral infection in the presence of antibody. In this way, the macrophage progeny from infected bone marrow cells is spreading the virus infection to new sites of the body. Because not all bone marrow macrophage-lineage cells are infected, only a small number of macrophages at peripheral sites are infected and produce virus. PRRSV capsids, consisting of ORF7 proteins only, can be formed in the absence of other viral proteins by, for instance infection of macrophages with a chimeric pseudorabies-ORF7 vector virus. The PRV virus was manipulated to contain ORF7 genetic information of PRRSV. After 18 hours post infection, the cytoplasm of infected cells contains large numbers of small, empty spherical structures with the size of PRRS virus nucleocapsids.